title: Characterization of the covalent binding of allyl isothiocyanate to β-lactoglobulin by fluorescence quenching, equilibrium measurement, and mass spectrometry.
abstract: Reversible binding of small compounds through hydrophobic interactions or hydrogen bonding to food proteins (e.g. milk proteins) is a thoroughly researched topic. In contrast, covalent interactions are not well characterized. Here, we report a rare form of positive-cooperativity-linear binding of allyl isothiocyanate with β-lactoglobulin, resulting in the cleavage of a disulfide bond of the protein. We compared three methods (i.e. fluorescence quenching, equilibrium dialysis, and headspace-water equilibrium) to characterize the binding kinetics and investigated the molecular binding by mass spectrometry. The methodologies used were found to be comparable and reproducible in the presence of high and low ligand concentrations for fluorescence quenching and equilibrium-based methods respectively.
authors.affiliation: a Institute of Human Nutrition and Food Science, Food Technology , Christian-Albrechts-Universität zu Kiel , Kiel , Germany .
authors.forename: Julia Katharina
authors.initials: JK
authors.lastname: Keppler
authors.forename: Tomas
authors.initials: T
authors.lastname: Koudelka
authors.forename: Kalpana
authors.initials: K
authors.lastname: Palani
authors.forename: Mayra Christina
authors.initials: MC
authors.lastname: Stuhldreier
authors.forename: Friedrich
authors.initials: F
authors.lastname: Temps
authors.forename: Andreas
authors.initials: A
authors.lastname: Tholey
authors.forename: Karin
authors.initials: K
authors.lastname: Schwarz
mesh_terms: D017879:Isothiocyanates / Q000737:chemistry*; D007782:Lactoglobulins / Q000737:chemistry*; D008024:Ligands; D013058:Mass Spectrometry; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
chemical_list: D017879:Isothiocyanates; D007782:Lactoglobulins; D008024:Ligands; C004471:allyl isothiocyanate
keywords: allyl isothiocyanate; covalent ligand binding; fluorescence quenching; tryptic digestion; β-lactoglobulin